Variable histone modifications at the A^vy metastable epiallele

Figures & data

Figure 1 The A^vy metastable epiallele. (A) The viable yellow agouti (A^vy) allele contains a contra-oriented IAP insertion within pseudoexon 1A (PS1A) of the Agouti gene. A cryptic promoter (short arrow labeled A^vy) drives constitutive ectopic Agouti expression. Transcription of the Agouti gene normally initiates from a developmentally regulated hair-cycle specific promoter in exon 2 (short arrow labeled A, a). (B) Genetically identical A^vy/a offspring representing the yellow and pseudoagouti coat color phenotypes are shown. * indicates 5′LTR of the A^vy IAP region and **represents non-IAP genomic PS1A region.

Figure 2 Chromatin precipitation for acetylated histones H3 and H4 in the 5′ LTR of the IAP and in PS1A. Binding activity was calculated as percent of pre-immunoprecipitated input DNA as represented by 2^−ΔC(t) X100. (A) DNA precipitated by H3 di-acetylation antibody is enriched in yellow versus pseudoagouti A^vy/a mice (p = 0.09; n = 6 per group) within the 5′ LTR of the IAP. (B) DNA precipitated by H4 di-acetylation antibody is enriched in yellow versus pseudoagouti A^vy/a mice (p = 0.08; n = 3 per group) within the 5′ LTR of the IAP. (C) DNA precipitated by H3 di-acetylation antibody does not vary by coat color within the PS1A genomic region (p = 0.4; n = 6 per group). (D) DNA precipitated by H4 di-acetylation antibody does not differ by coat color within the genomic PS1A region (p = 0.4; n = 3 per group). * indicates significance at the 0.09 level.

Figure 3 Chromatin precipitation for methylated histones in the 5′ LTR of the IAP and in PS1A. Binding activity was calculated as percent of pre-immunoprecipitated input DNA as represented by 2^−ΔC(t) X100. (A) DNA precipitated by H3K4 tri-methylation antibody is not enriched in yellow versus pseudoagouti A^vy/a mice (p = 0.7; n = 6 per group) within the 5′ LTR of the IAP. (B) DNA precipitated by H4K20 tri-methylation antibody is enriched in pseudoagouti compared to yellow A^vy/a mice (p = 0.01; n = 6 per group) within the 5′ LTR of the IAP. (C) DNA precipitated by H3K4 tri-methylation antibody does not vary by coat color within the PS1A genomic region (p = 0.9; n = 5 per group). (D) DNA precipitated by H4K20 tri-methylation antibody does not differ by coat color within the genomic PS1A region (p = 0.8; n = 5 per group). ** indicates significance at the 0.01 level.

Table 1 DNA methylation and Agouti (a) expression levels by histone and coat color status

	Histone mark				Liver DNA methylation					Liver (a) expression
	Liver histone trend	Mark type	Avg. (4–9)	Site 4	Site 5	Site 6	Site 7	Site 8	Site 9
Yellow	Enriched H3 and H4 di-acetylation	Activating	8.8%	10.5%	8.6%	8.2%	9.9%	7.2%	8.4%	600 Fold Increase
Pseudoagouti	Enriched H4K20 tri-methylation	Repressive	87%	91%	84.3%	88.7%	85.8%	81.7%	90.5%	Baseline

Table 2 PCR and pyrosequencing primers

Assay	Primer name	Primer sequence
A^vy IAP histone qPCR (251 bp)	A^vy_SY_F3	5′ AAA AAC AGA GTA CAA GTG GTC GTA 3′
	A^vy_SY_R3	5′ GAA ACA CAA CAT CTG GAT GGA 3′
A^vy PS1A histone qPCR (186 bp)	A^vy_SY_F5	5′ AGC ACC TGA ATT CAC ATT TG 3′
	A^vy_SY_R5	5′ TTT GCA CCA CAG TCG TTT T 3′
A^vy IAP CpG methylation PCR (312 bp)	IAPF3	5′ ATT TTT AGG AAA AGA GAG TAA GAA GTA AG 3′
	A^vy_Gen_R1	5′BIOTIN-CTA CAA AAA CTC AAA AAC TCA 3′
A^vy IAP pyrosequencing primer (CpG sites 4–5)	A^vy4–5	5′ GGA AAA ATA GAG TAT AAG TGG T 3′
A^vy IAP pyrosequencing primer (CpG sites 6–9)	CpG6F-short	5′ TAG AAT ATA GGA TGT TAG 3′
Agouti (a) expression qPCR (84 bp)	Agouti 1For	5′ TGG AGA TGA CAG GAG TCT GC 3′
	Agouti 1Rev	5′ TTC TTG TTC AGT GCC ACG AT 3′
Housekeeping gene expression qPCR (130 bp)	β-actin 1For	5′ GCT CCT TCG TTG CCG GTC CA 3′
	β-actin 1Rev	5′ ATA CAG CCC GGG GAG CAT CG 3′